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Genetic Control of Segmentation in Drosophila: Zygotic Gene Expression

by Dr. William Brook Department of Biochemistry and Molecular Biology, University of Calgary

We previously discussed the maternally localized factors that control segmentation in the Drosophila embryo. These factors influence the development of large portions of the embryo: the anterior and posterior halves. During early embryogenesis, these factors become distributed in concentration gradients. The nuclei in the pre-cellular embryo read these gradients, resulting in the subdivision of the embryo into domains of gap gene expression. A generalization is that gap gene expression patterns depend on activation by transcription factors encoded by maternal and gap genes and is refined by repression by other gap gene transcription factors ( A good review of this material can be found in (Hoch and Jäckle, 1993; Kornberg and Tabata, 1993).

How is the maternal gradient information transformed into gap gene expression patterns?

We have looked at hunchback already.

hunchback activation by Bicoid is an example of how a spatial pattern of gene expression could be established by a concentration threshold. (Remember: below a certain concentration Bicoid would no longer bind the promoter, so there would be no activation of hunchback in those nuclei).

We shall look at Krüppel as another example.

Krüppel is expressed as a stripe and so its regulation is more complicated but can be summarized as follows:

  • Bicoid activates Krüppel;
  • Hunchback activates Krüppel at low concentrations and represses Krüppel at high concentrations
  • Knirps represses Krüppel

What these genes do together is to define the expression zone of Krüppel from both the anterior and the posterior sides. Krüppel expression is activated by Bicoid and low levels of Hunchback throughout most of its region. However, the expression of Krüppel is repressed on the anterior and posterior sides by high levels of Hunchback and Knirps, respectively.

How is this studied at the molecular level? We shall look at the posterior border of Krüppel expression, which is controlled by competition between Bicoid and Knirps (Hoch et al., 1992).

Herbert Jäckle's group were studying the regulation of the gene Krüppel and had identified a730 bp fragment that could drive the expression of a lacZ reporter gene in the Krüppel pattern.

Using gel mobility shift assays, they found that both Bicoid and Knirps bound to this region. Furthermore, using DNase 1 footprinting they found that Knirps and Bicoid bound to the same 16 bp region within the 730 bp region. They made plasmid constructs that had seven copies of this 16-mer in front of a CAT gene and transfected it into Drosophila tissue culture cells

They found that adding a plasmid expressing bicoid increased the expression of CAT level in a dose-dependent manner. When they subsequently added a plasmid expressing the knirps gene, they found that they could reduce the level of CAT expression. This suggests that Bicoid and Knirps regulate the expression of Krüppel by competing for binding at this 16bp site. When Bicoid binds, Krüppel is activated. When Knirps binds, Krüppel is repressed.

Pair-rule genes (i.e. even-skipped, fushi tarazu)

The embryo is divided into large regions by the gap genes, which - along with the co-ordinate genes - activate transcription of the pair-rule genes in seven stripes of expression. The activation of the pair-rule genes in striped patterns by the maternal coordinate genes and gap genes is the first sign of segmentation in the embryo. Pair-rule gene expression patterns determine the position of the parasegments.

How is this co ordinated? It turns out there is a hierarchy within the pair-rule gene class. The primary pair-rule genes(even-skipped, hairy, and runt) are activated directly by the gap genes and they regulate the expression of the secondary pair-rule genes (fushi-tarazu, odd-skipped, odd-paired, paired, and others) .

How are the primary pair-rule genes regulated? It turns out that there is a separate enhancer controlling the expression of each stripe of gene expression in each of the primary pair-rule genes. The first evidence for this came from regulatory mutations that altered the transcription of specific stripes of expression. This was shown conclusively when the regulatory regions were analyzed using transgenes driving lacZ expression. It was discovered that there were regulatory elements for each stripe of expression. So upstream of even-skipped there are 7 independent regulatory elements - one for each stripe. The three primary pair-rule genes are expressed in 7 stripes each for a total of 21 over-lapping domains of gene expression. In principle, the way each of these stripes of expression is established is no different from the way the gap gene expression patterns are established through a combination of positive and negative inputs.

Let's look at what is happening with the expression of even-skipped in the second stripe from the anterior .

Bicoid and Hunchback activate even-skipped stripe 2 expression. even-skipped stripe 2 is repressed on the anterior side by the gap gene giant and on the posterior side by Krüppel.(Small et al., 1991)

Figure modified from Small et al., 1991.

Upstream of the even-skipped gene is a 430 bp enhancer element that controls just the expression of even-skipped in the stripe 2 region (it is aptly named the "stripe 2 enhancer"!). This enhancer has 12 known factor binding sites, including 6 activator and 6 repressor sites. The 6 activator sites include 5 Bicoid binding sites and one Hunchback site. There are 3 binding sites for each of Giant and Krüppel..

Diagram modified from (Arnosti et al., 1996)

even-skipped regulation is an example of the complex regulation of spatial gene expression by activation and repression at different control sites. As an example, in the stripe 1 domain, the stripe 1 enhancer activates even-skipped transcription and all others are inactive. In the domain between stripe 1 and 2, all seven enhancers are inactive. In the stripe 2 domain, the stripe 2 enhancer activates transcription and all others are inactive, etc.

First phase of segment polarity gene expression: pair-rule genes establish segment polarity gene expression patterns

The primary pair-rule gene expression patterns are established by the coordinate and gap genes and then refined through interactions with each other and with the secondary pair-rule genes. For example, even-skipped (eve) represses the expression of fushi-tarazu (ftz)leading to the expression of the two genes in complementary graded patterns of expression in alternating parasegments.

The pair-rule genes are already expressed in a periodic pattern, so it is easy to imagine how they establish the segment polarity gene expression in every parasegment. The expression patterns of the segment polarity genes engrailed (en) and wingless (wg) are established through positive and negative transcriptional regulation by the pair-rule genes. For example, the expression of en is activated by either Ftz or Eve in each parasegment, whereas wingless is repressed by Ftz or Eve in each parasegment. (How would these interactions be demonstrated, and how would it be shown that the interaction was direct or indirect?)

Other pair-rule genes also control wg and en expression. For example, paired and odd-paired are responsible for the activation of engrailed AND wingless in alternating stripes.

In-class exercise: expression patterns of wg and en in pair-rule mutants.

Parasegments and segments

These terms can be confusing. Parasegments and segments are different ways of subdividing the cells along the a/p embryonic axis . They are out of phase with each other. Parasegments correspond to domains of gene expression and to an early morphological feature seen before germ-band retraction, the parasegmental groove. At the time of the cellular blastoderm, the cells within each segment are morphologically indistinguishable from one another. The boundary between the parasegments is exactly between the engrailed and wingless expressing cells and is marked during gastrulation and early germ-band extension by a shallow groove. Beginning after the completion of germ band extension and during germ band retraction, the parasegmental grooves disappear at the same time as very deep grooves begin to arise halfway down the length of each parasegment - these will be the segmental boundaries.

Second phase of segment polarity gene expression: cell to cell signaling

The regulation of the segment polarity genes by the pair-rule genes is only the first stage of regulation. There are two problems that must be overcome. First, the expression of the coordinate, gap and pair-rule genes fades away and new mechanisms for regulating wingless and engrailed are required. Furthermore, cellularization of the embryo has occurred by this stage and it turns out that the mechanism for maintaining wingless and engrailed expression is based on cell to cell communication. This is why not all of the segment-polarity genes are transcription factors.

How are the patterns of wingless and engrailed maintained?

Genetic experiments showed that en and wg are required for each other's expression: Wg disappears in en mutant embryos; En disappears in wg embryos. This suggests that Wg and En maintain each other's expression. However, they are expressed in completely different cells, so cell- cell communication must occur.

engrailed encodes a homeodomain protein; wingless encodes a secreted peptide, a member of the WNT family. Wingless is secreted from the cells which make it. When the Wingless protein binds to its receptor on posterior cells, the signal is transduced to the nucleus and maintains the transcription of engrailed.

A second signal must be invoked to explain the maintenance of wingless expression by engrailed expression. Since Engrailed is a transcription factor, it cannot be directly responsible for the signal from the posterior engrailed expressing cells. wingless and engrailed expression are also lost in mutants for another segment polarity gene is called hedgehog. hedgehog is expressed in the same cells as engrailed and its expression is lost in wingless and engrailed mutants. These results imply that hedgehog is part of the genetic circuit linking wingless and engrailed expression. hedgehog encodes a secreted factor that is responsible for the signal from the engrailed expressing cells back to the wingless expressing cells. hedgehog binds to its receptor on the wingless expressing cells and this results in the maintenance of wingless transcription.

One of the clues that hedgehog was likely to be part of the circuit was that it had a phenotype that is very similar to that of wingless. A series of different genes mutate to the same phenotype (including cubitus interruptus, gooseberry, smoothened, fused, armadillo, disheveled, porcupine), and most of these have turned out to be part of either the wingless reception pathway or the hedgehog reception pathway.

Later in embryogenesis,wingless and engrailed expression become independent of one another, but the spatial patterns remain the same. So, the control of expression of the segment polarity genes wingless and engrailed goes through several distinct phases.

(See Gilbert 1997, Figures 14.25 and 14.26)

How do segment polarity genes control pattern in the segment?

The segment polarity gene control the pattern of cell differentiation within each segment. It has been proposed by several groups that wingless and hedgehog form concentration gradients that act as morphogens specifying different fates within the segment much in the same way that bicoid specifies fates within the anterior half of the embryo. For example, removing wingless function with a temperature-sensitive wingless allele after the expression of wingless and engrailed become expressed independently of one another results in a loss of the naked cuticle in each segment. Ubiquitous expression of Wingless in that same phase using a Heat shock-wingless transgene results in the transformation of the denticle belts into naked cuticle. This suggested that the level of Wingless is both necessary and sufficient for the fate of the cells in the naked cuticle.

LEGEND: Wingless and Engrailed expression in the embryo. The photograph shows a wild-type Drosophila cuticle with the approximate location of Wingless (blue) and Engrailed (yellow) expressing cells. The two diagrams show the realtionship between Wingless expressing cells, Engrailed expressing cells, and the differentiation of cuticle markers in normal embryos (middle) and embryos compromised for Wingless activity (bottom)
(Figure courtesy of Dr. Tim Heslip.)

Lawrence and collaborators (1996) examined the effects of manipulating the concentration of Wingless. Embryos that were mutant for both wingless and engrailed lack virtually any evidence of segmentation. By adding back different levels of Wingless using a transgene capable of producing different levels of wingless mRNA, they were able to demonstrate a concentration dependence for Wingless in specifying the fate of cells in the segment. For example, by adding back high levels of Wingless, they were able to produce segments consisting of cuticular elements normally found near the source of Wingless in the segment. Progressively lower levels produced cuticular structures normally found further from the wingless stripe in each segment. (See DiNardo et al., 1994 for a review of the regulation of segment polarity genes.)

Heemskerk and Dinardo (1994) demonstrated that Hedgehog had a concentration dependent effect on the fate of cells in the segmented dorsal epidermis. They accomplished this by either removing Hedgehog activity using a temperature sensitive mutant or by increasing the levels of Hedgehog using a transgene that used the inducible heat shock promoter driving hedgehog transcription.


  • The expression of the gap genes is regulated by a combination of positive and negative transcriptional regulation mediated by transcription factors encoded by the maternal coordinate genes and the gap genes.
  • Krüppel expression is controlled by Bicoid, Hunchback and Knirps. Competition between Knirps and Bicoid binding at the same target sequence determines the posterior border of Krüppel expression.
  • The primary pair-rule genes are regulated directly by the coordinate and gap genes. Each stripe of primary pair-rule gene expression is regulated by a separate enhancer element.
  • The pair-rule genes establish the periodic expression patterns of wingless and engrailed expression
  • Wingless and Engrailed maintain each other's expression as part of a regulatory circuit involving cell-cell interactions.
  • Wingless may specify fate in the epidermis in a concentration dependent manner.

Learning Objectives

  • Outline the role of Bicoid in the regulation of the gap gene hunchback and in the regulation of the gap gene Krüppel.
  • There is a conflict between your text and the results from (Hoch et al., 1992). The text says that Bicoid is a repressor of Krüppel but Hoch et al say that it activates the expression of Krüppel. Explain the conflict. (Hint: the claim that Bicoid is a repressor of Krüppel expression is based on the anterior expansion of Krüppel transcription in embryos that lack bicoid function.
  • Describe the types of experiments necessary to make the diagram of the even-skipped stripe 2 enhancer shown above.
  • Why must the segment polarity genes have more than one mode of regulation?
  • Why is it thought that Wingless may specify fate in a concentration-dependent manner?

Digging Deeper:

See Peifer and Bejsovec, 1992, and DiNardo et al., 1994) for reviews of the regulation of segment polarity genes.

Reviews* and References

Arnosti, D. N., Barolo, S., Levine, M., and Small, S. (1996). The eve stripe 2 enhancer employs multiple modes of transcriptional synergy. Development 122, 205-14.

*DiNardo, S., Heemskerk, J., Dougan, S., and O'Farrell, P. H. (1994). The making of a maggot: patterning the Drosophila embryonic epidermis. Curr Opin Genet Dev 4, 529-34.

Heemskerk, J., and DiNardo, S. (1994). Drosophila hedgehog acts as a morphogen in cellular patterning. Cell 76, 449-60.

Hoch, M., Gerwin, N., Taubert, H., and Jackle, H. (1992). Competition for overlapping sites in the regulatory region of the Drosophila gene Kruppel. Science 256, 94-7.

*Hoch, M., and Jackle, H. (1993). Transcriptional regulation and spatial patterning in Drosophila. Curr Opin Genet Dev 3, 566-73.

*Kornberg, T. B., and Tabata, T. (1993). Segmentation of the Drosophila embryo. Curr Opin Genet Dev 3, 585-94.

Lawrence, P.A., Sanson, B., and Vincent, J.P. (1996). Compartments, wingless and engrailed: Patterning the ventral epidermis of Drosophila embryos. Development 122, 4095-4103.

Small, S., Kraut, R., Hoey, T., Warrior, R., and Levine, M. (1991). Transcriptional regulation of a pair-rule stripe in Drosophila. Gene Develop 5, 827-839

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Last revised Monday, November 16, 1998